Development of DNA Polymerase ? Inhibitors for Precision Medicine in BRCA Deficient Cancers DNA polymerase theta (Pol?) is an error-prone polymerase that is highly upregulated in breast and ovarian tumors. Over expression of Pol? is correlated with a poor clinical outcome for breast cancer patients. However, the role of Pol? in breast and ovarian cancer progression and survival has not been studied until recently. For instance, new ground breaking studies have identified a major role for Pol? in error-prone DNA repair and tie this function to the survival of cancers mutated in BRCA1 or BRCA2 (BRCA1/2) tumor suppressor genes. For example, we have discovered that Pol? promotes an error-prone DNA repair pathway called microhomology-mediated end-joining (MMEJ)??also known as alternative end-joining (a-EJ)??which causes chromosome rearrangements and genome instability, a common phenotype associated with BRCA deficient cancers. Moreover, other related studies have shown that the DNA synthesis activity of Pol? is essential for MMEJ, and that targeted inactivation of the polymerase domain stalls the proliferation of BRCA deficient cells, but has no effect in normal cells. These studies strongly suggest that pharmacological inhibition of Pol? will selectively kill BRCA deficient cells. Given that cells defective in BRCA1/2 or other major HR factors combined with a deficiency in Pol??a backup DNA repair factor?are synthetic lethal, we hypothesize that chemical inhibitors of Pol? will cause specific killing of BRCA deficient cancer cells while sparing normal cells. We have utilized the specialized function of Pol? in MMEJ to develop a novel high-throughput screening assay. Since MMEJ activity is unique to Pol? our MMEJ based screening assay may preferentially identify specific inhibitors of this function of the polymerase which is important for cancer cell survival. We anticipate that the development of Pol? inhibitors will enable inactivation of the polymerase in cells and thus cause selective killing of BRCA deficient cancer cells. We will identify and test Pol? inhibitors by developing the following aims: 1. To conduct high- throughput screening for Pol? inhibitors; 2. To validate Pol? inhibitors and test their specificity for the polymerase; 3. To identify Pol? inhibitors that selectively kill BRCA deficient cells. The successful development of Pol? inhibitors is likely to lead to a new form of precision medicine for BRCA deficient cancers.